The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) was one of the latest to evolve and one of the last to be identified, partly because they are not strong oncogenic drivers. TAM RTKs are ectopically expressed or overexpressed in a wide variety of human cancers in which they provide tumor cells with a survival advantage. TAM RTKs also play a critical role at the interface of the innate and adaptive immune response. The TAM RTK expressed in individual tumors or in different tumor types are not necessarily redundant; each may connect to downstream survival or motility signaling in slightly different ways.
TAM (Tyro3-Axl-Mer) RTK activation mechanisms are unique, as maximal stimulation involves both an extracellular lipid moiety and a bridging protein ligand. The ligands are γ-carboxylated proteins that bind to the receptor with their carboxy-terminal domain and to the lipid phosphatidylserine (PtdSer) with their amino terminus. The first such ligand, growth arrest-specific protein 6 (GAS6), was purified from conditioned media from normal lung and endothelial cell lines, and binds to all three TAM RTKs. A second γ-carboxylated protein, vitamin K-dependent protein S (PROS1), binds only to MERTK and TYRO3. In the body, PtdSer is abundant but only available to activate TAM receptors when externalized on apoptotic cell membranes, aggregating platelets, exosomes and invading virus envelopes.
TAM receptor and ligand overexpression have been shown in a wide range of solid and hematological tumors, and correlate with poor prognosis in a variety of tumor types and their signals and promote survival, chemoresistance, motility and invasion. In addition, their role in diminishing the innate immune response makes their inhibition a novel mechanism for reversing the immunosuppressive tumor microenvironment.
Regarding MerTK, the best-studied TAM RTK function is the role of MERTK in efferocytosis—the process by which apoptotic material is cleared by both monocyte-derived and epithelial cells. In macrophages, MERTK activation leads to engulfment of apoptotic material and suppression of the inflammatory cytokine response. During apoptotic cell ingestion, MERTK suppresses the M1 macrophage pro-inflammatory cytokine response (involving interleukin-12 (IL-12), IL-6 and tumor necrosis factor (TNF)), partly by diminishing nuclear factor-KB (NF-κB) signaling, and also enhances M2 macrophage anti-inflammatory cytokine production. MERTK signaling also alters macrophage gene expression, which suppresses inflammatory cytokine production and polarizes the macrophage towards a wound-healing, anti-inflammatory M2 phenotype. Thus, MERTK functions in macrophages to promote the rapid clearance of self antigens, to repair injured tissue and to suppress inflammation. When MERTK is eliminated or inhibited, apoptotic cells languish, which allows the proliferation of non-tolerant B cells, enhanced CD4+ T helper cells and the release of inflammatory cytokines. In tumor-associated macrophages, MERTK inhibition might therefore lead to enhanced antitumor immunity.
The tumor-associated macrophage and its less well-studied counterpart, the monocytoid myeloid-derived suppressor cell (MDSC), are derived from monocyte lineage cells that express little or no MERTK. However, in tissues, differentiated subsets induce the expression of MERTK. One major MERTK-expressing macrophage subtype, M2c, is differentiated in response to macrophage colony-stimulating factor (M-CSF; also known as CSF1). In the tumor microenvironment, continued MERTK activation by dying cells suppresses macrophage NF-κB signaling and the downstream induction of inflammatory cytokines (for example, IL-12 and IFNγ), and MERTK-mediated increases in IL-10 and GAS6 ensue. Inhibition of MerTK can alter the tumor microenvironment to a pro-inflammatory, tumor suppressive environment reducing the immunosuppressive nature of MDSCs.
Additionally, activated T cells induce the expression of PROS1 and externalize limited PtdSer patches on T cell membranes. This T cell-based ligand complex directly contacts innate immune cells, activating MERTK and turning down inflammatory cytokine production. Inhibition could lead to an immune-modulating effect promoting an M1 innate immune response, fueling a Th1 T cell response. The latter would supplement immune checkpoint (anti-CTLA4 or PD1) and tumor vaccine strategies.
Regarding Axl, dendritic cells, which are more dependent on AXL than on MERTK, provide feedback that helps to terminate inflammatory Toll-like receptor (TLR) signaling. In these antigen-presenting cells (APCs), TLR signaling results in activation of STAT1, which in turn induces AXL mRNA. AXL functions together with the type I interferon (IFN) receptor to increase suppressor of cytokine signaling 1 (SOCS1) and SOCS3 expression, which helps to terminate inflammatory TLR signaling. Axl inhibition could aid in restoring inflammatory TLR signaling.
Most patients with solid tumors die of metastatic disease rather than from the primary tumor. AXL in particular has been implicated in metastasis in multiple tumor types. First, AXL has a role in normal directed motility in the nervous system during the migration of gonadotropin-releasing hormone (GNRH)+ neurons to the hypothalamus. Second, in patient samples and cell lines, AXL expression correlates with migration and metastasis. Third, metastasis often requires epithelial-to-mesenchymal transition (EMT), which is facilitated by AXL. Canonical EMT-inducing gene products TWIST, SNAIL (also known as SNAI1) and SLUG (also known as SNAI2) are induced by AXL overexpression or through GAS6 stimulation. TWIST and SNAIL can also stimulate AXL expression, reinforcing EMT. Axl inhibition has been shown to reduce metastatic proliferation.
AXL also plays a well-established role in resistance to targeted therapeutics and examples of acquired resistance are currently limited to AXL. AXL is upregulated in imatinib-resistant CML and gastrointestinal stromal tumor (GIST) cell lines and tumor samples, and siRNA-mediated knockdown of AXL restored imatinib sensitivity to resistant cell lines. Similarly, AXL is induced in lapatinib-resistant HER2 (also known as ERBB2)-positive breast cancer cell lines, and AXL inhibition restored lapatinib sensitivity. AXL has been associated with acquired resistance to epidermal growth factor receptor (EGFR) TKIs and therapeutic antibodies in triple-negative breast cancer and head and neck cancer cell lines, as well as with resistance to inhibitors targeting other kinases, including fibroblast growth factor receptor (FGFR), anaplastic lymphoma kinase (ALK) and insulin-like growth factor 1 receptor (IGF1R). AXL is upregulated in NSCLC cell lines and xenografts that are resistant to EGFR TKIs and antibody drugs (cetuximab and erlotinib), and it is induced in 20% of matched tumor samples taken from patients with NSCLC after development of resistance to erlotinib (an EGFR TKI). The broad range of cancers studied, implicate AXL in drug resistance and suggest that AXL inhibition may have widely applicable utility and could re-sensitize tumors to targeted therapies.
Regarding MerTK and Axl dual inhibitors, the normal roles of MERTK and AXL in preventing or terminating innate immune-mediated inflammation and natural killer (NK) cell responses are subverted in the tumor microenvironment. MERTK and AXL decrease NK cell antitumor activity, which paradoxically allows increased metastases.
In addition, in solid tumors overexpression of AXL and MerTK promote chemoresistance. Therefore, targeting AXL allowed for increased potency of small molecule MerTK inhibitors, both on its ability to decrease the activation of MERTK and downstream effectors, as well its ability to decrease proliferation and colony formation. The cooperative relationship between MERTK and AXL, and coordinated regulation of expression has been demonstrated. Specifically, inhibition of either receptor increases expression of the other receptor. Additionally, MERTK and AXL are capable of physical interaction, suggesting that heterodimerization between MERTK and AXL may be a relevant mechanism of dual receptor activation. Targeting these two receptors concurrently provided synergistic decreases in oncogenic signaling, cell proliferation and colony formation.
Dual inhibition of MERTK and AXL may be a rational combination strategy that may have clinical utility against NSCLC and other solid tumors.
Regarding Pan TAM family inhibitors, much less is known about Tyro3 because it has been understudied. Using a 3-D spheroid assay to study the effect on motility, migration and invasion using a range of solid tumor cell lines, Pan TAM inhibition was very effective at inhibiting invasion and migration in the collagen culture.